The Radical SAM enzyme NirJ catalyzes the removal of two propionate side chains during heme d1 biosynthesis. (15th November 2017)

• Record Type: Journal Article
• Authors: Boss, Linda; Oehme, Ramona; Billig, Susan; Birkemeyer, Claudia; Layer, Gunhild
• Is Part Of: FEBS journal. Volume 284:Number 24(2017)
• Page Start: 4314
• Page End: 4327
• Publication Date: 2017-11-15
• Subjects: denitrification -- heme d1 biosynthesis -- NirJ -- Radical SAM enzyme -- tetrapyrrole; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.14307
• Languages: English
• ISSNs: 1742-464X

Abstract

Abstract : Heme d 1 is a modified tetrapyrrole playing an important role in denitrification by acting as the catalytically essential cofactor in the cytochrome cd 1 nitrite reductase of many denitrifying bacteria. In the course of heme d 1 biosynthesis, the two propionate side chains on pyrrole rings A and B of the intermediate 12, 18‐didecarboxysiroheme are removed from the tetrapyrrole macrocycle. In the final heme d 1 molecule, the propionate groups are replaced by two keto functions. Although it was speculated that the Radical S ‐adenosyl‐l ‐methionine (SAM) enzyme NirJ might be responsible for the removal of the propionate groups and introduction of the keto functions, this has not been shown experimentally, so far. Here, we demonstrate that NirJ is a Radical SAM enzyme carrying two iron–sulfur clusters. While the N‐terminal [4Fe‐4S] cluster is essential for the initial SAM cleavage reaction, it is not required for substrate binding. NirJ tightly binds its substrate 12, 18‐didecarboxysiroheme and, thus, can be purified in complex with the substrate. By using the purified NirJ/substrate complex in an in vitro enzyme activity assay, we show that NirJ indeed catalyzes the removal of the two propionate side chains under simultaneous SAM cleavage. However, under the reaction conditions employed, no keto group formation is observed indicating that an additional cofactor or enzyme is needed for this reaction. Abstract : The heme d 1 biosynthesis enzyme NirJ belongs to the Radical SAM protein family and binds two iron–sulfur clusters. One of the clusters is required for the typical SAM cleavage reaction. Overall NirJ catalyzes the removal of two propionate side chains from the substrate 12, 18‐didecarboxysiroheme.